Purified hepatitis c virus envelope proteins for diagnostic and therapeutic use

Abstract

The present invention relates to a method for purifying recombinant HCV single or specific oligomeric envelope proteins selected from the group consisting of E1 and/or E1/E2 characterized in that upon lysing the transformed host cells to isolate the recombinantly expressed protein a disulphide bond cleavage or reduction step is carried out with a disulphide bond cleavage agent. The present invention also relates to a composition isolated by such a method. The present invention also relates to the diagnostic and therapeutic application of these compositions. Furthermore, the invention relates to the use of HCV E1 protein and peptides for prognosing and monitoring the clinical effectiveness and/or clinical outcome of HCV treatment.

1 . A recombinant vector comprising a vector sequence and an: appropriate eukaryotic or viral promoter sequence operably linked to a nucleotide sequence comprising an HCV signal sequence attached before a sequence encoding an HCV envelope E2 protein starting at amino acid position 384 and ending in the region between amino acid positions 600 and 820 and wherein said HCV envelope E2 protein is produced by recombinant expression in lower eukaryotic or mammalian host cells.  
     
     
         2 . The recombinant vector according to  claim 1 , with said nucleotide sequence being characterized further in that it ends at any of amino acid positions 623, 650, 673, 710, 715, 720, 746 or 809.  
     
     
         3 . The recombinant vector according to  claim 1 , wherein said nucleotide sequence is comprising a sequence encoding an HCV signal sequence starting at amino acid position 347 or 364 and ending at amino acid position 383 and a sequence encoding an HCV envelope E2 protein starting at amino acid position 384.  
     
     
         4 . The recombinant vector according to  claim 1 , with said nucleotide sequence being characterized further in that a 5′-terminal ATG codon and a 3′-terminal stop codon have been added to it.  
     
     
         5 . The recombinant vector according to  claim 1 , with said nucleotide sequence being characterized further in that a factor Xa cleavage site and/or 3 to 10, histidine codons have been added.  
     
     
         6 . The recombinant vector according to  claim 5 , wherein said histidine codons are added 3′-terminally to the coding region.  
     
     
         7 . The recombinant vector according to  claim 1 , with said HCV E2 encoding sequence being chosen from the group as represented in SEQ ID NO 35, 37, 39, 41, 43, and 45.  
     
     
         8 . The recombinant vector according to  claim 1 , with said nucleotide sequence encoding an HCV protein spanning amino acid positions 384-746, 384-809, 384-673, 347-746, 347-809, 347-673, 347-683, 364-673, 364-746 or 384-673.  
     
     
         9 . Recombinant nucleic acid comprising any of the HCV E2 encoding sequences as represented in SEQ ID NO 35, 37, 39, 41, 43, 45.  
     
     
         10 . Recombinant nucleic acid encoding an HCV E2 protein spanning amino acid positions 384-746, 384-809, 384-673, 347-746, 347-809, 347-673, 347-683, 364-673, 364-746 or 384-673.  
     
     
         11 . Recombinant vector according to  claim 1 , further characterized in that at least one of the glycosylation sites present in said HCV protein has been removed by site-directed mutagenesis of the nucleotide sequence encoding a glycosylation site.  
     
     
         12 . An isolated host cell transformed with at least one recombinant vector according to  claim 1 .  
     
     
         13 . A composition comprising a recombinant vector according to  claim 1.